drLumi: An open-source package to manage data, calibrate, and conduct quality control of multiplex bead-based immunoassays data analysis

Abstract

Multiplex bead-based immunoassays are used to measure concentrations of several analytes simultaneously. These assays include control standard curves (SC) to reduce between-plate variability and normalize quantitation of analytes of biological samples. Suboptimal calibration might result in large random error and decreased number of samples with analyte concentrations within the limits of quantification. Suboptimal calibration may be a consequence of poor fitness of the functions used for the SC, the treatment of the background noise and the method used to estimate the limits of quantification. Currently assessment of fitness of curves is largely dependent on operator and that may add additional error. Moreover, there is no software to automate data managing and quality control. In this article we present a R package, drLumi, with functions for managing data, calibrating assays and performing quality control. To optimize the assay the package implements: i) three dose-response functions, ii) four approaches for treating background noise and iii) three methods for estimating limits of quantifications. Other implemented functions are focused on the quality control of the fitted standard curve: detection of outliers, estimation of the confidence or prediction interval, and estimation of summary statistics. With demonstration purpose, we apply the software to 30 cytokines, chemokines and growth factors measured in a multiplex bead-based immunoassay in a study aiming to measure correlates of risk or protection from malaria of the RTS,S malaria vaccine nested in the Phase 3 randomized controlled trial of this vaccine.

Modulation of innate immune responses at birth by prenatal malaria exposure and association with malaria risk during the first year of life

“…Samples were acquired on a Luminex® 100/200™ instrument using Xponent 3.1 software. Median fluorescent intensity (MFI) data was analyzed using the drLumi 0.1.2 R package [ 42 ], in which concentration of each analyte was determined by interpolating the MFI to a standard curve (plotted using a 5- or 4-parameter logistic function) of twofold 16 serial dilutions prepared from a reference sample provided by the manufacturer. The limits of quantification (lower, LLOQ and upper, ULOQ) for each analyte and plate were obtained applying the 20% coefficient of variation method [ 43 – 45 ] in drLumi.…”

A Comparative Analysis of Multipotent Mesenchymal Stromal Cells derived from Different Sources, with a Focus on Neuroregenerative Potential

“…All experiments were repeated 3 times with different donors. The data were obtained using a Luminex ® 200 ™ Instrument (EMD Millipore, Billerica, MA, USA) operated by xPonent software 3.1.871.0 (Luminex Corporation, Austin, TX, USA) and the data were processed by drLumi package 67 in R statistical environment 68 . The median fluorescence intensities (MFI) of cytokine and growth factor standard dilutions were used for standard curve fitting using 5-parameter logistic regression (SSL5) with 4-parameter logistic regression (SSL4) as a fallback in case the SSL5 model did not converge.…”

Advances in Proteomic Techniques for Cytokine Analysis: Focus on Melanoma Research

“…( A ) Typical calibration curve covers 3–4 LOGs of cytokine concentration; ( B ) for some cytokines, additional data points at the lower end of the curve enable quantification of low cytokine concentration in samples and expansion of quantification over 4 orders of magnitude in cytokine concentration. Standard curves are calculated by drLumi package [ 132 ] in R statistical environment, full line shows standard curve fit to calibration standards (blue diamonds), dotted lines show prediction interval. Data points of real samples of experimental pig model body fluids are shown as orange circles.…”